sonic hedgehog Flashcards
How do secreting cells alter Shh before it is secreted?
N-terminal carbohydrate added in the golgi body followed by N-terminal palmitoylation by Skinny
How is Shh released from cells? what restricts movement? How does it bind to the receiving cell?
Hh released from cells by Dispatched (multimer, heparan sulphate proteoglycan, permits EC transport)
Hh interacting protein (Hip) can restrict movement
iHog/Boi facilitate binding to receiving cell. (iHog/Boi mutants show defects in neural differentation and phenocopy Smo mutants > essential for Shh pathway)
Described the receptor for Shh and explain what happens in resting state i.e. when No Shh is bound
Receptore is Patched.
12 span TM protein
In absence of Hh, Patched inhibits Smoothened (7 span TM protein)
what happens when Shh binds to Patched receptor? (step one)
hh binding prevents Ptc inhibition of Smo, Smo is phosphorylated
What happens after Smo is phosphorylated? (next step)
Smo activates Gli transcription factors
Where is GliA usually?
Held in the cytoplasm by suppressor of fused (SuFu), also mediates slimb association and proteosome proccessing to repressor form GliR
Slimb = drosophila protein, targets Ci for ubiquitylation and protesome processing to repressor form CiR.
Explain the negative feedback loop seen in Ptch/hh interaction
Ptc represses smo in the absence of Hh
Hh induces Gli activation
Ptc is a transcriptional target of Gli
More Ptc is inserted into the membrane, repressing Smo
What is hh involved in in Drosophila?
Larval body segment development
How does hh act in a paracrine fashion in drosophila?
Engrailed/Wingless/Ci
During body segment formation, stripes of cells synthesis Engrailed and also express Hh.
Hh cannot move far from where it is released so only activates a thins tripe of cells adjacent to Engrailed expressing cells
Only cells to one side of Engrailed-expressing cells are competent to respond to hh/Ptc interaction
Cells with activated Ptc receptors synthesise wingless
Wingless binds to Ci (Gli equivalent), results in incr wingless transcription of cells adj to stripe of Hh-prod cells
Wingless acts as an EC signal, patterns adjacent rows of cells by activating Frizzled (Wnt)
Wingless acts on engrailed-expressing cell to stabilise engrailed expression
Why is hh signalling important in drosophila?
Reciprocal signalling of hh & wingless stabilises the boundary between parasegments.
Effects of wingless & Hh on other stripes of cells in each segment establishes positional code, accounts for anatomical features along the AP axis
Shh mutants= body like a hedhehog
Why are cilia important for hedgehog signalling?
All the key components of hh signalling are enriched in cilia - Gli TFs, Smo & Ptc1
IFT is req. for Gli activation, esp. in limb development –> IFT mutants = polydactyly
BETH ADD MORE (LOOK AT DISSO)
Where is Shh produced during neurulation? what does it cause?
Explain role of Shh in neural tube
Produced by the notochor, causes differentiation of ventral cells of Neural tube, important in creating gradients in neural tube formation
results in movement of axons towards ventral surface of developing spinal cord
Bending of NP occurs at MH & DLHP
MHP is induced by notocord signalling - sole site of bending in upper spinal neural plate
DLHP - SHH signalling from notocord inhibits DLHP formation at upper spinal level
As wave of spinal neurulation passes down the neuraxis, strength of Shh sinalling from notocord decreases, allowing DLHP formation to ‘break through’ at lower spinal levels
DL binding is negatively regulated by Shh - DLHP formation in lower spine can be inhib by local shh release
Supported by mice studies - Shh null mutant = DHLPs at every level and NT closes correctly
Shh overexpression can produce NTDs - inhibit DL binding in places essential for correct NT closure
What does notocord Shh signalling induce in somitogenesis?
Induces ventral somite to differentiate into sclerotome
Ectopic expression found markers e.g. Pax1
It is thought that Shh works with other Hh ligands, what do deleterious mutations in either/both ligands cause?
Wnt1,3,4 +Shh –> myogenesis
Shh KO = cyclopia/NTD/somite/foregut patterning defects
shh + Ihh work togerher, double KO = embryonically lethal –> heart morphogenesis malformation
Which experiments formed the basis of the morphogen hypothesis?
Saunders & Gasseling (1969) on chick limb bud found that a diffusible factor was involved in development, later shown to be Hh
Where is Shh produced during limb bud formation?
Shh is produced by ZPA (small region of tissue at posterior margin of the limb)
Why is the ZPA significant during limb development?
Signalling within ZPA leads to development of the limb along AP axis
wnt7a is required for Shh in ZPA
Why is the AER significant during limb development?
Part of ectoderm, secretes FGF8 which induces Shh and maintains the limb gradient dorsally
How is shh involved in digit patterning?
Current model in murine embryos= concentration and time of Shh exposure determines which digits form
Works in an autocrine fashion (digits 5,4 and some of 3 arise from cells expressing shh)
Digit 5: longest expression, highest conc of Shh
Digit 4: Less time and conc of Shh (some diffused from 5)
Digit 3: Lesser so compared to 4&5
Digits 5,4&3 form in absence of Dispatched - autocrine signalling takes place, however Dispatched is required for EC diffusion to aid formation of other digits
Digit 2: receives EC Shh from digit 3 (has dispatched) (shh cant move far)
Digit 1: doesn’t require shh - default programme of limb bud cells
How is hh signalling involved in eye development?
Shh involved in early stages of eye development
Eye develops from a population of cells from ANTERIOR NEURAL PLATE
Eye fields originally located centrally, separates into two fields located centrally.
Eye fields separate into two lateral optic vesicles
Shh secreted from the midline is required for separation of the eye field into 2 lateral optic vesicles
shh mutant mice = cyclopia
holoprosencephaly in humans
How is hh signalling involved in ear development?
Shh antagonizes Wnt protein from roof plate which refines and maintains dorsoventral axial patterning in the ear.
Shh directly promotes the development of spiral ganglion neuron during neurogenesis. After Shh signaling pathway is activated, Shh indirectly upregulates Ngn1 expression > regulating neurogenic patterning of inner ear.
Shh regulates the differentiation of hair cells - influences the cell cycle of cochlea progenitor cells
Basal-to-apical wave of Shh decline ensures the normal development pattern of hair cells
Experiments/ Evidence for shh involvement in NTDs
homozygous extra-toes mouse has a cranial neurulation defect. Mutant for Gli3 which negatively regulates shh signalling. Gli 1 & 2 mediate Shh > mutations do not lead to NTDs
Ptc-1 (shh off) mutation > ligand independent consitutive activation of shh signallling. Ptc-1 null mice = severe NTDs (same as Shh overexpression)
PKA = heteromeric enzyme, inhibits Shh signalling - decr PKA dose by 75% leads to a high freq of spinal defects >overrtim of shh inhibi DL binding of neural folds, leading to spina bifida and exencephaly
There is an indication that Shh is neg reg DLHP formation at high spinal levels to ventralise closing NT & prevent dorsally located cells from differentiating in a direction necessary for DLHP formation … mutant studies?
Two other mutants: open-brain & zic2 = failure of NT closure in both brain & lower spine
Opb - disrupts rab23 gene (encodes neg reg of shh signalling)
Opb homozygous = dorsal & dorsolateral cell fates are not specified in developing NT, indicated by a lack of wnt3a & msx1 genes
Shh regulation of neural fold bending is a mechanism operated at every level of the body axis
PKA = heteromeric enzyme, inhibits Shh signalling - decr PKA dose by 75% leads to a high freq of spinal defects >overrtim of shh inhibi DL binding of neural folds, leading to spina bifida and exencephaly
How is shh involved in NS boundary formation?
ZLI = Zona limitans intrathalamica, at border of thalamus and prethalamus within the diencephalon
ZLI aries as a broad wedge of cells which narrows, marked by absence of Lfng expression (modulator of Notch signalling)
ZLI is a compartment - does not mix with adjacent thalamus
ZLI expresses Shh - Shh also expressed in floorplate for hindbrain and spinal cord forming neurons
shh induces region-specific gene expression in thalamus and prethalamus
Distinct gene expression due to different competence to respond to Shh
How is Shh involved in cortical development?
Fate of neural stem/progenitor cells is determined by their position
Type of neuron and/or glial cell generated by the NSC/NPC is determined by the position of the cell in the developing brain, controlled by expression of BMP and Shh
How is Shh involved in heart development?
shh & FGF are involved in compact layer formation in the compaction & trabeculation stage of heart development
Shh & Ihh work together - double KO is embryonically lethal due to heart morphogenesis malformation
